The main objective of this proposal will be to elucidate the cellular origin of renal erythropoietin and the mechanisms that regulate its secretion. The kinetics of erythropoietin production will be studied in animals exposed to hypoxia or injected with cobalt chloride. Erythropoietin will be measured by bio and radioimmunoassays and the sites of production will be identified by monospecific antibodies. Isolated perfused kidneys, renal cell fractions, primary renal cultured cells and established cell lines will be studied physiologically and biochemically for their production of erythropoietin. Erythropoietin will be labelled endogenously with radioactive aminoacids and erythropoietin will be identified by immunoprecipitation followed by electrophoresis and fluorography of the precipitated material. Messenger-RNA will be isolated from whole kidneys and isolated cells and translated in vitro. Inhibitors of RNA synthesis, protein synthesis inhibitors and inhibitors of glycosylation will be utilized in order to determine the renal synthesis and secretion of erythropoietin. Specific c-DNA Epo probes will be utilized for identification of Epo mRNA extracted from tissue homogenates and also for in-situ hybridization in tissue slices. The elucidation of the mechanism that controls erythropoietin synthesis and secretion will increase our understanding of the pathophysiology of the anemia of chronic renal disease and secondary polycythemias.